Proteinase-activated receptors (PARs) are a family of G protein–coupled receptors that are widely expressed on neurons and glial cells in the nervous system (1). PARs are activated through proteolytic cleavage of their extracellular NH2 terminus. The proteolytic cleavage unmasks a “tethered ligand” that binds intramolecularly to the receptor and initiates a signal transduction event (2). Among the four PARs identified to date, PAR1, PAR3, and PAR4 can be activated by thrombin, whereas trypsin and mast cell tryptase can activate PAR2 (2, 3). Signaling through different heterotrimeric G proteins, PARs can affect various cellular functions in the nervous system, including neural cell proliferation, gene transcription, differentiation, and survival (4, 5). The role of PAR2, which is widely distributed throughout the nervous system, has been principally investigated in the peripheral nervous system, where it is known to play major roles in injury, inflammation, neuronal signaling, and nociception (6, 7). PAR2 is also known to be expressed on neurons and astrocytes in rodent and human central nervous systems (CNS), and several studies have implicated it in the pathogenesis of ischemia and neurodegeneration (8–10).

Multiple sclerosis (MS) is a common immune-mediated neurological disorder, which is histopathologically characterized by infiltration of the CNS with inflammatory leukocytes followed by demyelination and axonal loss (11–13). It is generally accepted that an autoimmune response directed against components of myelin is the chief pathogenic event during MS (14). The exact mechanisms leading to the generation of autoimmune responses in MS are not fully known, although components of both the adaptive and innate immune systems are involved (15, 16). Experimental autoimmune encephalomyelitis (EAE) is a widely studied animal model for MS, which recapitulates many of the clinical and neuropathologic aspects of MS (17). EAE can be induced by immunization of genetically susceptible animals with different antigenic components of CNS myelin, including myelin basic protein (MBP), proteolipid protein, or myelin oligodendrocyte glycoprotein (MOG). Immunization leads to the generation of myelin-reactive T cells in the periphery, which then migrate into the CNS and initiate autoimmune inflammation. Although considered largely a T cell–mediated disease, there is increasing evidence for the involvement of other immune cells, including activated macrophages and microglia in both the initiation as well as the effector phases of the immune response associated with the EAE and MS pathogenesis (16, 18). Herein, the expression levels and the cell types expressing PAR2 were investigated in the CNS of MS and control patients. Using EAE as an animal model of MS, we explored the role of PAR2 activation on astrocytes and monocytoid cells. Experiments were performed to evaluate the impact of PAR2-deficient signaling on the generation and responsiveness of myelin-reactive T cells and the severity of MOG-induced EAE.

Results

PAR2 expression is increased in the white matter during MS and EAE

PAR2 has been shown to be widely expressed on neurons and astrocytes in the CNS (19, 20). To investigate the role of PAR2 in the neuroinflammatory process associated with MS, we examined PAR2 transcript levels in CNS white matter from MS and non-MS patients. RT-PCR analysis showed significantly higher PAR2 transcript levels in the white matter of MS compared with non-MS patients (Fig. 1 A
). Transcript levels of trypsinogen, a potential PAR2-activating proteinase, were not different between MS and non-MS CNS tissues (Fig. 1 B). Immunohistochemical staining showed that PAR2 immunoreactivity was markedly enhanced in MS (Fig. 1 C) compared with non-MS white matter (Fig. 1 D). In MS CNS tissues, PAR2 immunoreactivity was chiefly detected on infiltrating perivascular cells together with parenchymal glial cells in the areas of active demyelination (Fig. 1 E). Indeed, PAR2 immunoreactivity was colocalized with CD45 leukocyte/macrophage marker in perivascular cells (Fig. 1 E) and with glial fibrillary acidic protein (GFAP) astrocytic marker in parenchymal cells (Fig. 1 F). PAR2 immunoreactivity was present on neurons in the gray matter with no obvious differences between groups (not depicted). Previous studies of blood-derived mononuclear cells have reported that monocytes but not lymphocytes express PAR2, and moreover, the expression levels increase upon differentiation to macrophages (21, 22). We also examined PAR2 expression in PBMCs from healthy human volunteers. Flow cytometric analysis showed PAR2 expression on the monocyte population (Fig. 1 G), whereas expression on lymphocytes was minimal (Fig. 1 H). Treatment of PBMC cultures with phytohemagglutinin for 24 h resulted in a marked up-regulation of PAR2 immunoreactivity on monocytes (Fig. 1 G), but the PAR2 immunoreactivity on the lymphocyte population was unaffected (Fig. 1 H). Thus, these observations indicated that monocytes rather than lymphocytes were the chief cell type expressing PAR2 in both blood and brain, in support of previous studies (21, 22).

Given that the EAE animal model recapitulates many of the neuropathological and clinical features of MS, together with similar underlying pathogenic mechanisms, we next examined PAR2 expression in the CNS of mice with EAE. Interestingly, RT-PCR analysis showed significantly higher levels for PAR2 mRNA in the CNS of animals with EAE compared with healthy control mice (Fig. 2 A
). Similar to the MS CNS samples, PAR2 immunoreactivity was detectable in the white matter of EAE animals, where it was localized chiefly on GFAP-immunopositive astrocytes (Fig. 2 C, top) and ionized calcium binding adaptor protein (Iba-1)–immunopositive macrophage/microglia (Fig. 2 C, bottom). Hence, these findings indicated that PAR2 expression was up-regulated on both glial cells and infiltrating leukocytes during MS/EAE-associated neuroinflammation.

PAR2 activation mediates oligodendrocyte toxicity

Acute demyelinating lesions in MS are believed to be generated by infiltrating leukocytes and activated microglia that destroy myelin in the presence of autoreactive T cells (26, 27). However, oligodendrocyte injury and apoptosis, even in the absence of a substantial inflammatory reaction, have also been reported to be a principal pathological feature in newly forming lesions (28, 29). We investigated the indirect effects of PAR2 activation on macrophages and astrocytes in terms of oligodendrocyte viability. Supernatants from cultured murine macrophages or astrocytes, treated with the PAR2-activating peptide (PAR2 AP) SLIGRL-NH2 or the mutant inactive peptide (mAP) LSIGRL-NH2, were applied to oligodendrocyte cultures for 24 h, and the surface area of GalC-immunopositive oligodendrocytes as well as the number of cells with processes were quantified. Interestingly, supernatants of PAR2 AP–treated, but not mAP- or mock-treated macrophages, caused a marked reduction in the surface area of exposed oligodendrocytes, leaving a higher number of cells with fewer or no processes (Fig. 4 A
). Quantification of the oligodendrocyte area and the number of cells with processes showed significant reductions in cultures treated with the supernatants from PAR2 AP–stimulated macrophages compared with mock- or mAP-treated macrophages (Fig. 4 B). Conversely, supernatants of PAR2 AP–treated astrocytes did not affect oligodendrocyte morphology and surface area (Fig. 4 C). Gene expression studies revealed a significant induction of TNF-α (Fig. 4 D), iNOS (Fig. 4 E), IL-6 (Fig. 4 F), and IL-12p40 (Fig. 4 G) transcript levels in PAR2 AP–treated macrophages compared with mAP- or mock-treated cells. In addition, IL-1β, IFN-inducible protein 10, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1α transcripts were increased significantly in PAR2 AP–treated macrophages, but not in mAP- or mock-treated cells (not depicted). The same effects were not observed for PAR2 AP–treated astrocytes (not depicted). Thus, these findings revealed that direct activation of PAR2 on macrophages caused oligodendrocyte injury associated with inflammatory gene expression, which might contribute to demyelination in MS.

Given the higher expression of PAR2 in human MS CNS white matter and its contribution to inflammatory gene expression and oligodendrocyte injury, we next investigated the potential role of PAR2 in EAE, as an animal model of MS, using PAR2 WT and KO mice. We examined MOG-induced EAE in 10–12-wk-old PAR2 KO mice compared with their WT littermate controls and corresponding healthy (intact) controls. Neuropathological examination of the CNS of WT animals with EAE showed markedly enhanced immunoreactivity for macrophage/microglial marker, Iba-1, compared with PAR2 KO EAE animals and the nonimmunized intact group (Fig. 5 A
, first column). Iba-1–immunopositive cells showed hypertrophy and were more frequently detected in WT and PAR2 KO EAE groups compared with the quiescent morphology observed in the nonimmunized intact group. Quantitative analysis showed a significantly higher number of Iba-1–immunopositive cells in the PAR2 WT EAE group compared with PAR2 KO EAE animals (Fig. 5 B). MBP immunoreactivity showed disrupted integrity of the myelin in both PAR2 WT and KO EAE groups with much more severe demyelination in WT animals (Fig. 5 A, second column), as indicated by quantitative analysis of MBP-immunopositive areas of the white matter (Fig. 5 C). Indeed, myelin loss was closely associated with macrophage infiltration/microglial activation, as indicated by merged MBP–Iba-1 immunoreactivity (Fig. 5 A, third column). CD3+ T cells were also detectable in spinal cords after EAE induction (Fig. 5 A, fourth column), with the WT EAE group showing a significantly higher number of positive cells compared with KO EAE animals (Fig. 5 D). Silver staining followed by counting of the axonal number also showed a significant reduction in the number of axons in the PAR2 WT EAE group compared with the KO EAE and control groups (Fig. 5 E). Of note, PAR2 WT and KO nonimmunized (intact) animals did not differ in terms of neuropathological features and therefore were pooled for these quantitative analyses. Overall, these findings indicated that deficient PAR2 signaling reduced neuroinflammation with greater preservation of myelin and proximate axons in the CNS during MOG-induced EAE.

The generation of myelin-reactive T cells in the peripheral immune system with their subsequent infiltration of the CNS has been considered a key event in EAE and MS pathogenesis (11). To examine the proliferative and cytokine response of T cells in MOG-immunized PAR2 WT and KO animals, we measured [3H]thymidine incorporation and intracellular IFN-γ immunoreactivity by FACS in lymphocytes cocultured with MOG-loaded irradiated splenocytes. Proliferation of T cells measured by [3H]thymidine incorporation was significantly higher in WT animals compared with their PAR2 KO littermates (Fig. 6 A
). Intracellular cytokine assays showed a significant increase in IFN-γ reactivity of CD3:CD4 but not CD3:CD8 immunopositive lymphocytes of WT animals compared with PAR-2 KO littermates (Fig. 6, B and C). These observations disclosed that despite the absence of PAR2 on lymphocytes, there was a significant difference in T cell reactivity at the level of proliferative and cytokine responses after MOG immunization of WT and KO animals.

Enhanced host immune responses are apparent within the CNS during MS and EAE (13). Gene expression studies of WT and KO animals showed significantly higher levels of TNF-α (Fig. 7 A
), iNOS (Fig. 7 B), IL-6 (Fig. 7 C), and IFN-γ (Fig. 7 D) mRNA in the CNS of WT animals with EAE compared with their PAR-2 KO littermates. TNF-α, iNOS, IL-6, and IFN-γ mRNA was detectable in PAR2 WT and KO animals without EAE (intact) with no differ-ence between the two groups (Fig. 7, A–D). Together with differences in the neuropathological findings and T cell reactivity, these observations emphasized the role of PAR2-deficient signaling in mitigating MOG-induced autoim- mune processes.

Discussion

This study highlights previously unrecognized interactions between PAR2 expression and induction of neuroinflammation during MS/EAE, thereby disclosing new therapeutic opportunities for CNS autoimmune demyelinating disorders. We have demonstrated that PAR2 expression is enhanced in the CNS white matter during MS and EAE in which it is chiefly expressed on perivascular macrophages and astrocytes (Fig. 9
). PAR2 WT and deficient macrophages and astrocytes exhibited differential inflammatory gene expression, with PAR2 activation on macrophages leading to the induction of proinflammatory cytokines and chemokines adversely affecting oligodendrocyte viability (Fig. 9). Indeed, absent PAR2 signaling suppressed EAE disease severity, which was associated with decreased T cell reactivity and neuroinflammation.

PAR2's neuroinflammatory actions in the CNS during MS/EAE. Activation of PAR2 on macrophages and astrocytes results in the release of proinflammatory (NO, TNF-α, IL-1β, and IL-6) and oligodendrocytotoxic molecules together with chemokines that contribute to the recruitment of myelin-reactive T lymphocytes into the CNS during MS/EAE.

It is widely recognized that autoreactive T cells infiltrating the white matter during MS initiate demyelination through the release of toxins together with recruiting and activating monocytoid cells (16). However, an alternative view holds that oligodendrocyte death, perhaps due to apoptosis, together with microglial activation in the absence of marked lymphocyte infiltration might herald newly forming demyelinating lesions (29). Indeed, several studies indicate that innate immune mechanisms are potent determinants of MS and EAE disease onset and severity (30–33). Extensive oligodendrocyte death likely overwhelms normal mechanisms of dead cell clearance, prompting a T cell–mediated inflammatory response that arises from the release of myelin antigens (29). In this study, we observed less T cell reactivity and infiltration after EAE induction in PAR2-deficient mice, which may partly explain the milder disease severity in this group of animals compared with their WT littermates. Nonetheless, we also found that PAR2 activation on macrophages led to the production of soluble cytotoxic factors, which diminished oligodendrocyte viability. These two observations could be considered reciprocal mechanisms by which PAR2 expression affects the underlying pathogenic processes in EAE, likely reflecting the interaction of the adaptive and innate immune systems. However, it is also plausible that PAR2 expression and activation on monocytoid cells (unlike astrocytes) contributes directly to oligodendrocyte injury (34), which could prime a subsequent myelin-specific T cell response, as mentioned above.

A recent study has reported a neuroprotective role for neuronal PAR2 in experimental stroke (35). Moreover, our group showed a neuroprotective role for neuronal PAR2 expression and activation in the context of HIV-induced dementia, a neurodegenerative disorder caused by the HIV infection of brain (24). Herein, up-regulation and activation of PAR2 on monocytoid cells contributed to the neuroinflammatory/degenerative process in EAE. Of interest, PAR2 expression is up-regulated on monocytes upon differentiation to macrophages (22), and its activation leads to the production of proinflammatory cytokines IL-1β, IL-6, and IL-8 (21). A concomitant localized increase in tryptic serine proteinases, which act as potential activators of PAR2, may accentuate PAR2-mediated effects in the context of MS/EAE neuroinflammation. Indeed, there is also a report describing the role of PAR2 in dendritic cell development (36), indicating that dendritic cells do not spontaneously develop from the bone marrow cells of PAR2-deficient mice after IL-4/GM-CSF treatment, despite differentiating to mature dendritic cells in the presence of inflammatory mediators (36). In our hands, treatment of bone marrow cells from WT and PAR2 KO animals with IL-4/GM-CSF led to the generation of morphologically indistinguishable dendritic cells, which matured normally after LPS treatment (not depicted). Nonetheless, given the administration of adjuvants upon MOG immunization with the resulting immune activation, it is unlikely that differences in antigen presentation exert major effects on the ensuing autoimmune processes in the present studies.

Although many studies have indicated proinflammatory roles for PAR2 in respiratory, gastrointestinal, musculoskeletal, skin, and peripheral nervous systems (23, 37–42), PAR2 agonists have also been shown to be beneficial in some mouse models of mucosal inflammation (43, 44). These studies underscore the different roles played by PAR2 based on the individual cell type and specific animal model or disease context. Elucidation of mechanisms leading to altered receptor expression and differential downstream effects, together with identifying specific activating proteinases, will permit the development of new therapeutic strategies for modulating detrimental effects of inflammation in the nervous system.

Real-time RT-PCR.

Cultured cells, human brain (frontal lobe white matter), or lumbar spinal cords from mice were homogenized in TRIzol (Invitrogen) according to the manufacturer's guidelines. Total RNA was isolated and dissolved in diethylpyrocarbonate-treated water, and 1 μg RNA was used for the synthesis of complementary DNA and subsequent PCR as described previously (48). Primer sequences were as follows: human GAPDH, 5′ primer: 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′,3′primer: 5′-CGGAGTCAACGGATTTGGTCG-3′; human PAR2, 5′ primer: 5′-CTGGCCATTGGGGTCTTTCTGTTC-3′, 3′ primer: 5′-GGCCCTCTTCCTTTTCTTCTCTGA-3′; human trypsinogen, 5′ primer: 5′-TCAGCGAACAGTGGGTGGTATCAG-3′, 3′ primer: 5′-GAGGGGCGGTGGGCAGAG-3′; mouse PAR2, 5′ primer: 5′-TGGCCATTGGAGTCTTCCTGTT-3′, 3′ primer: 5′-TAGCCCTCTGCCTTTTCTTCTC-3′; and mouse trypsinogen, 5′ primer: 5′-ATCTCTGGCTGGGGCAACACTC-3′, 3′ primer: 5′-CTAGGAAGCCAGCACAGACCA T-3′. Mouse trypsinogen primers were designed based on the mouse mesotrypsin (mouse serine protease 3, Prss3) sequence. The other mouse oligonucleotide primer sequences have been reported previously (54). Semiquantitative analysis was performed by monitoring in real time the increase of fluorescence of the SYBR green dye on an i-Cycler (Bio-Rad Laboratories) as reported previously (48). All data were normalized against the GAPDH mRNA levels and expressed as fold increases relative to controls ± SE.

Induction and assessment of EAE.

10–12-wk-old female PAR2 homozygous KO mice (50) and littermate homozygous WT controls were used for EAE induction. Age-matched female mice were injected subcutaneously with 50 μg MOG (MOG35–55 peptide; prepared by the Peptide Synthesis Facility, University of Calgary) emulsified in 100 μl of complete Freund's adjuvant (Difco Laboratories; reference 47). Animals received intraperitoneal injections of pertussis toxin (0.3 μg; List Biological Laboratories) at the same time as MOG immunization and 48 h later. Vehicle-treated animals were only injected with complete Freund adjuvant and pertussis toxin. Animals were assessed daily for EAE severity for 30 d using a 0–5 rating scale as reported previously (55). Animals were killed by cardiac puncture under ketamine/xylazine anesthesia. Spinal cords were removed and fixed in 4% neutral buffered formalin before paraffin embedding or lysed in TRIzol for RNA extraction. All experiments were approved by the University of Calgary Animal Care Committee.

Histological analysis.

Formalin-fixed spinal cords of EAE or vehicle-treated animals were embedded in paraffin before sectioning (56). 4-μm sections from lumbar spinal cords were stained with Bielschowsky's silver impregnation method (33). Five randomly chosen fields in each animal's white matter were scanned and photographed using a microscope (Axioskop2; Carl Zeiss MicroImaging, Inc.) and the Spot imaging system (Diagnostic Instruments). The number of silver-positive axons was quantified in square millimeters using Scion Image software (Scion Corporation).

T cell proliferation and flow cytometric analysis.

Splenocytes isolated from the spleens of nonimmunized animals by density separation over Ficoll-hypaque were γ irradiated, suspended at a density of 2 × 106 cells/ml, and incubated with 40 μg/ml MOG35–55 peptide for 30 min (57). Splenocytes incubated with vehicle were used as control. Draining lymph nodes were isolated from MOG-immunized PAR2 WT and KO animals 7 d after immunization. Lymph nodes were homogenized in PBS, and lymphocytes isolated from dissociated lymph nodes were washed and suspended at a density of 2 × 106 cells/ml. Splenocytes and lymphocytes were plated 1:1 in 96-well U-bottom microtiter plates. Cells were incubated at 37°C for 48 h before adding 1 μCi [3H]thymidine (GE Healthcare) to each well. Cells were harvested after 24 h and counted on a liquid scintillation counter. Intracellular cytokine assay was performed using a cytofix/cytoperm kit according to the manufacturer's guidelines (BD Biosciences). In brief, Golgistop protein transport inhibitor was added to splenocyte/lymphocyte cocultures 48 h after plating. Cells were harvested after 12 h and immunostained using PerCP- labeled anti–mouse CD3 (1:50), FITC-labeled anti–mouse CD4 (1:50), and PE-labeled anti–IFN-γ (1:50) monoclonal antibodies. All monoclonal antibodies were purchased from BD Biosciences. For PAR2 immunodetection, cultured PBMCs were stained with B5 rabbit anti-PAR2 antibody followed by Alexa 488–conjugated goat anti–rabbit secondary antibody. FACS analysis was performed on a FACSCalibur apparatus using CELLQuest software (Becton Dickinson).

Acknowledgments

This work was supported by the Multiple Sclerosis Society of Canada (MSSC) and Canadian Institutes for Health Research (CIHR) operating grants (N. Vergnolle, M.D. Hollenberg, and C. Power) and a CIHR Proteinase and Inflammation Network Group grant. S. Tsutsui is an MSSC Fellow. N. Vergnolle is an Alberta Heritage Foundation for Medical Research Scholar/CIHR New Investigator. C. Power holds a Canada Research Chair (Tier 1) in Neurological Infection and Immunity.

Riches, D.W., and G.A. Underwood. 1991. Expression of interferon-beta during the triggering phase of macrophage cytocidal activation. Evidence for an autocrine/paracrine role in the regulation of this state. J. Biol. Chem.266:24785–24792.